The goal of this proposal is to determine the molecular mechanisms by which sexual development is controlled in the opportunistic human fungal pathogen Cryptococcus neoformans. Sexual development results in the production of spores that can be dispersed into the environment. C. neoformans is thought to infect humans from environmental sources via a respiratory route in which fungal spores are inhaled into the alveoli of the lungs. Once inhaled, C. neoformans can disseminate to the central nervous system and cause a fatal form of meningitis. Understanding the biology of sexual development and spore production in C. neoformans promises to provide information useful in the prevention and/or treatment of cryptococcal disease. This study is also of general interest because opportunistic fungi are emerging pathogens: understanding basic fungal biology sets the stage for the development of more effective methods of preventing and treating fungal disease in general. In C. neoformans sexual development is controlled by the Sex Inducer genes, SXI1a and SXI2a, but the mechanism by which they act is unknown. The experiments outlined in this proposal are designed to elucidate the mechanism by which SXI1a and SXI2a regulate gene expression to drive sexual differentiation. Using genetic, biochemical, bioinformatic, and cell biological approaches we will 1) map the regulatory circuit controlled by Sxi1a and Sxi2a by identifying and characterizing their downstream targets, 2) establish the properties of the Sxi1a and Sxi2a proteins required to control sexual development by evaluating the relationships between their structures and functions, and 3) investigate the cellular processes required for sexual development with a specific focus on dikaryotic filamentation. The diverse approaches presented here will elucidate the early stages of sexual development and lend new insights into how infectious particle production occurs in C. neoformans.